ecent evidence demonstrates that exercise induces delayed cardioprotection similar to that of ischemic preconditioning (PC), ia mechanisms that are presently unknown. Unlike ischemic or pharmacologic PC, exercise PC is triggered by a physiologic itimulus and thus would appear to be a natural means for achieving cardioprotection. The overall objective of this proposal is .0 elucidate the molecular mechanisms .underlying the newly-discovered phenomenon of exercise PC. Our fundamental lypothesis is that exercise-induced release of NO (via eNOS) activates a signal transduction cascade that includes PKCe, Src/Lck, and multiple transcription factors and culminates in the upregulation of iNOS, which then mediates the protection, either singularly or in conjunction with eNOS. A broad multidisciplinary approach will be used that will combine diverse echniques (integrative physiology, protein chemistry, mass spectrometry, biochemistry, cell biology, molecular biology, gene .argeting, and transgenesis) and will integrate genetic information at the molecular level with biochemical information at the jrotein structure level and physiological information at the whole animal level. Unequivocal evidence for or against an Dbligatory role of three specific kinases (PKCe, Src,.Lck) in exercise PC will be provided by the use of a novel dominant negative PKCe transgenic mouse line and Src and Lck knockout mice, this will enable us to achieve, for the first time, (inase-specific modulation of PKCe. Src.and Lck during exercise. The role of PKCs in initiating exercise PC will be onclusivelv established by determining the effects of specific transgenic inhibition of this isozyme. The kinase-specific activity of all seven Src PTKs expressed in the mouse heart (Fyn, Fgr, Yes, Src, Lyn, Lck, and Blk) will be directly measured at serial times after exercise PC. The role of Src PTKs in triggering versus mediating exercise PC'will be discerned by comparing inhibition of these kinases on days 1 and 2 (during the exercise stimulus) versus day 3 (during coronary occlusion). Targeted disruption of the Src and Lck gene will be employed to conclusively establish the specific function of individual PTKs in the PC protection. The transcription factors responsible for exercise PC will be systematically interrogated by using mice with targeted genetic ablation of each of the main factors known to bind to the iNOS gene (IRF-1, TNF-a, STAT1, CREB, AP-1, IL-2, and IL-6). The specific NOS isoforms responsible for initiating as well as mediating exercise PC will be conclusively identified by targeted gene disruption of eNOS, iNOS, and nNOS. The post-translational modulation of iNOS 24 h after exercise will be elucidated by identifying the precise phosphorylation site(s) on iNOS with HPLC coupled- electrospray ionization mass spectrometry. Finally, the role of Src PTKs in the post-translational modulation of iNOS will be established by measuring iNOS activity and tyrosine phosphorylation in the absence and presence of Src PTK inhibitors. This proposal should produce important new insights into the molecular mechanisms whereby the heart adapts not only to physical stress but also to stress in general. Elucidation of the mechanism of exercise PC may have important therapeutic implications.